Unique self-assembled poly-amidoamine polymers and their eletrochemical reactivity

ABSTRACT

Synthesis of novel and unique PAMAM (poly-amidoamine) polymers. PAMAM polymers can be grown by systematic alternation between ethylenediamine (EDA) and methacrylate. By taking advantage of the alternating terminal ends, successive generations G1 and G0.5 were combined under acidic conditions with Pluronic P123 as a liquid-crystal template. The resulting polymer was imaged with TEM and the product was circular and amorphous of no characteristic size ranging between about 5 nm to about 600 nm, with remarkable electrochemical activity unseen in any of the generations of PAMAM. Applications of this electroactive poly-amidoamine organic polymer include use as a new electron transfer reagent for amperometric biosensors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/316,468, filed on Dec. 5, 2016, now U.S. Pat. No. 10,323,008,which was a 371 application of PCT/US2015/034495 filed Jun. 5, 2015,which claims priority to U.S. Provisional Patent Application No.62/008,923 filed on Jun. 6, 2014. All of the foregoing are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Biomarker electrochemical testing constantly demands greater degrees ofsensitivity and specificity. Specifically, medical conditions thatrequire point of care testing like diabetes are increasing in prevalencein the United States. With increasing demand, the market requires highquality tests that cost less to produce. One approach to this problem isutilizing highly ordered nanomaterials. The use of mesoporous carbonelectrodes has shown to produce a remarkable increase in the sensitivityand the specificity of electrochemical testing.

Nanoparticles and nano-scale structures have become a cornerstone ofpoint of care testing. Nanoparticles can be conjugated to enzymes toalter the frequency at which they are best detected, while mesoporousmaterials have been used as electrodes for detecting small moleculeslike glucose, uric acid, lactate, and other similar molecules. Carbonelectrodes are cheap and effective ways of producing electrochemicalsensors for biochemical detection. Alteration of the structure of thecarbon in these electrodes allows for more accurate detection.

Creating tailored mesoporous materials is a step forward to achieving ahigher degree of sensitivity and level of detection. It has beendemonstrated that the type of material used to detect has a significanteffect on the performance of the sensor. These results show thatmesoporous silica outperformed a mesoporous carbon electrode for thedetection of ascorbic acid, uric acid, and xanthine. Although mesoporouscarbon out performs silica for the detection of lactate and glucose, itis not sufficient to merely control the physical features of themesoporous materials, they must be tailored specifically to the analytetrying to be detected. Therefore, there is a need for creating tailoredmaterials that may provide a higher degree of sensitivity and level ofdetection than is conventionally available.

SUMMARY OF THE INVENTION

This invention relates to a polymer capable of conducting electronsthrough solution, in the presence of a metal working electrode, bymobilizing metal ions into the solution.

Some embodiments provide a chemical composition that includes:

In which 1 includes (CH₂CH₂)[N(CH₂CH₂CONHCH₂CH₂N*)₂]₂; and 2 includes(CH₂CH₂)[N(CH₂CH₂CONHCH₂CH₂N(CH₂CH₂CO*)₂)₂]₂.The “*” indicates the point of attachment. Other embodiments provide achemical composition that includes a polymer capable of conducting anelectric current through a solution, the polymer including at least oneof molecule 1 (as described above) and one of molecule 2 (as describedabove) and configured to perform in-vivo electrochemical biomarkerdetection.

Further embodiments provide a method of performing biomarker detectionincluding synthesizing a polymer capable of conducting an electriccurrent through a solution, the polymer including at least one polyamidepolymer and configured to perform in-vivo electrochemical biomarkerdetection. The method further includes performing in-vivoelectrochemical biomarker detection using the polymer.

These and other aspects will be apparent upon reference to the followingdetailed description and figures. To that end, any patent and otherdocuments cited herein are hereby incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of self-assembly of a mesoporous materialfrom a polymer composition.

FIG. 2A depicts TEM (transmission electron microscopy) images of EPOP(Electroactive Poly(amidoamine) Organic Polymers) particles understandard conditions.

FIG. 2B depicts TEM images of EPOP particles under standard conditions.

FIG. 3 depicts CV data that illustrate the electrical activity of EPOP,with inset electrical activity of Poly(amidoamine), or PAMAM. CV of EPOPis shown with average peaks around −0.55V and 0.25V. The peaks shift toa small degree based on the concentration of EPOP in the solution. Whencombined with other electro active species the CV of EPOP changes inunique patterns based on the other electro active substance.

FIG. 4 depicts CV data that illustrate electrochemical activity of EPOPas compared to FERRI.

FIG. 5 depicts data that illustrate a battery AMP-it. EPOP is shown toperform differently than a classical capacitor. The minimum activatingcurrent in FIG. 5 is about −0.4V. The material is sensitive to pH, whichhas effects on the magnitude and the duration of discharge.

FIG. 6 depicts data that illustrate that very acidic solutionssignificantly reduce the function of EPOP, while mildly acidic solutionshave a moderating effect.

FIG. 7 illustrates data that demonstrate that pH is not the only factthat has an effect on the way EPOP behave, reversal voltage effects thepeak current −0.55V −0.4V −0.65V, length of charge time 10 extraseconds, the amount of sample 504 increased to 1004, double charging thesample.

FIG. 8 depicts data illustrating electroactive properties of a polymercomposition on an electrode system using carbon, carbon, and copper.

FIG. 9 depicts data illustrating electroactive properties of a polymercomposition achieved on an electrode system using nickle, carbon, andcopper.

FIG. 10 depicts data illustrating electrochemical detection of glucosemolecules using a polymer composition as a detection sensor.

FIG. 11 illustrates the change in calculated time constants of thesolutions of an electroactive compound under different conditions.Average time constants: 1, 1.42E+01; 2, 2.45E+01; 3, 4.93E+00; 4,4.65E+00; 5, 2.07E+02; 6, 5.56E+00. Total Current: 1, 6.91E-03; 2,7.50E-03; 3, 1.14E-02; 4, 8.46E-03; 5, 6.23E-03; 6, 5.99E-03. Capacity:1, 1.55E-02; 2, 1.73E-02; 3, 2.99E-02; 4, 2.02E-02; 5, 2.72E-02; 6,1.29E-02.

FIG. 12 illustrates a linear standard curve based on the data fromglucose detection.

FIG. 13A illustrates a polymer composition of the invention.

FIG. 13B illustrates a polymer composition of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure relates to regents for electrochemical detection, energystorage, and alternative energy sources. More specifically, thisinvention relates to polymers capable of conducting electrons throughsolution, in the presence of a metal working electrode, by mobilizingmetal ions into the solution. This invention has been used to create ametal polymer electrolytic cell, capable of operating at high currentsat low voltages for the purposes of electrochemical detections, energystorage, and other alternative energy technologies.

In an embodiment, the synthesis of a novel and unique PAMAM(poly-amidoamine) polymer is disclosed. PAMAM polymers can be grown bysystematic alternation between ethylenediamine (EDA) and methacrylate.By taking advantage of the alternating terminal ends, successivegenerations G1 and G0.5 were combined under acidic conditions withPluronic P123 as a liquid-crystal template. The resulting polymer wasimaged with TEM and the product was circular and amorphous of nocharacteristic size ranging between about 5 nm to about 600 nm. Thoughgenerally disordered, this polymer demonstrates remarkableelectrochemical activity substantially unseen in any of the generationsof PAMAM. This electroactive poly-amidoamine organic polymer can be usedas a new electron transfer reagent for amperometric biosensors.

Nano-scale materials provide three critical features that effect how asensor performs: filtration, encapsulation, and surface area. The smallpore size in these materials prevents large interfering structures fromreaching the surface of the electrode. Other fields of research likemass spectrometry also exploit this filtration effect; nanoporous ormesoporous materials allow small molecules to pass through where theyinteract with antibodies before being ionized. This filtrationeffectively removes a significant source of noise and error in wholeblood or serum samples. Concordantly the small diameters can also beused to encapsulate enzymes and antibodies. Encapsulation increases thenumber of enzymes that are at the electrode surface, while decreasingartifact from Brownian motion in solution. The features of encapsulationhave been utilized to create a sensor for lactate. These features alonedemonstrate a significant need for being able to control the structuralfeatures of materials on a nano-scale.

Polyamidoamine polymers (PAMAM) have been extensively studied for theirdendrimeric properties. They can be created at room temperature, understandard conditions, to produce high product yields. Starburst and othergenerations of PAMAM have been studied as drug delivery vehicles, and avariety of other applications including electrochemical testing. PAMAMdendrimers can be synthesized in a variety of ways, with interchangeablelinking units modifying the properties based on the desired applicationand design. PAMAM can be used like a molecular level Napoleonicinterchangeable parts system, where each successive generation providesa specific tailored attribute to the behavior of the dendrimer. It isthis high degree substitutability that makes PAMAM dendrimers an idealpolymer to study the synthesis of mesoporous structures.

PAMAM's stepwise synthesis provides an ideal experimental material toprovide a proof of concept for utilizing the liquid-crystal templatemethod for creating specifically tailored nano-structured materials. Thetwo reactions in PAMAM synthesis happen under mild conditions that arecompatible with a templating solution. As shown in FIG. 1, successivegenerations of PAMAM dendrimers have terminal functional groups thatreadily react with each other linking the two generations togetheraround the template. Concentric symmetry allows for growth in alldirections simultaneously that can be controlled by the number ofterminal groups present on the generation of PAMAM.

Example 1

Materials:

All chemicals were purchased from Sigma-Aldrich. Ethylenediamine 99%,Methyl Acrylate 99%, Methanol Anhydrous 99%, Pluronic P123, n-butanol.Silica Gel, NaCl 99%, HCl solution (1M).

Methods:

Synthesis of G −0.5

2 grams (1.8 ml) of EDA was dissolved into 100 ml of methanol and cooledin an ice bath. 13.6 mmol (1.112 ml) of methyl acrylate was addeddropwise under stirring. The solution was allowed to react for 168 hoursat room temperature. The excess methyl acrylate was removed undervacuum. The remaining viscous liquid was purified via silica gel columnchromatography with a 10:1 dichloromethane/methanol eluant.

Synthesis of G0

17.85 grams (16.065 ml) of EDA was dissolved into 10 ml of methanol andcooled to −30° C. in dry ice. 1.37 g of G −0.5 PAMAM was dissolved into2.5 ml of methanol and cooled to −30° C. in dry ice. The dendrimersolution was added gradually to the EDA solution without significantchange in temperature. The solution was allowed to warm to roomtemperature and react for 168 hours at room temperature (25° C.). 100 mlof n-butanol was added to the solution, and the excess EDA was distilledoff with the n-butanol.

Synthesis of G0.5

1.37 grams of G0 PAMAM was dissolved into 20 ml of methanol and cooledin an ice bath. 23.2 mmol (1.897 ml) of methyl acrylate was addeddropwise under stirring. The solution was allowed to react for 96 hoursat room temperature and 24 hours at 45° C. The excess methyl acrylatewas removed under vacuum. The remaining viscous liquid was purified viasilica gel column chromatography with a 10:1 dichloromethane/methanoleluant.

Synthesis of MC-A

1.85 grams of NaCl and 0.336 g of Pluronic 123 were dissolved into 2.8ml of 1M HCL. To this solution 0.752 grams of G0.5 PAMAM and 0.188 gramsof G0 PAMAM were added successively under vigorous stirring at roomtemperature. The mixture was stirred at room temperature for 24 hours.The solution was then incubated at 45° C. for 24 hours. All solidmaterial was filtered out, and an additional (3 ml) of G0 was addedunder vigorous stirring to the solution. This solution was stored at 0°C. for 72 hours. The amber solution was dried via vacuum distillation,and orange crystals precipitated from the solution. The surfactant coreswere removed with a 25:1 acetone/HCl mixture for 2 days at 40° C. Theextraction product was filtered off and washed with acetone, leaving awaxy orange coagulation of crystals.

Synthesis of MC-B

1.85 grams of NaCl was dissolved into 2.8 ml of 1M HCL, any excesscrystals were filtered out via vacuum filtration. 0.336 g of Pluronic123 was then added to the solution and was allowed to mix untilhomogeneous. To this solution 0.6 grams of G0.5 PAMAM was added andallowed to mix completely, subsequently 0.3 grams of G0 PAMAM were addeddropwise under vigorous stirring at room temperature (25° C.). Themixture was stirred at room temperature (25° C.) for 24 hours. Thesolution was then incubated at 45° C. for 24 hours. The liquid phase wasfiltered, the amber solution was dried via vacuum distillation, andorange crystals were precipitated from the solution. The surfactant corewas removed with a 25:1 acetone/HCl mixture for 2 days at 40° C. Theextraction product was filtered off and washed with acetone, leaving awaxy orange coagulation of crystals.

CV

Using the mass of 100 mM, 0.032 g/mL ferricyanide as a reference,solutions ranging from 0.01 g/mL to 0.08 g/mL of MC-A was prepared inPBS. The sample was tested on a screen-printed three lead carbonelectrode. The sample was run from 1.5 to −1.5 for six segments toobtain a CV.

Results:

The resulting polymer from the synthesis is a orange-amber soft solid,stable at room temperature (25° C.). Two synthesis lots MC-A and MC-Bdiffered only by minor changes in the final synthesis step. Completelydried MC-A physically was more like a dry crystal, though soft andeasily compressed into other shapes. MC-B retained a waxy, almost gellike, soft consistency and was lighter in color. Both MC-A and MC-B areequally soluble in water, though MC-A seems to dissolve slightly fasterthen MC-B.

As shown in FIGS. 2A and 2B, TEM imaging of MC-B shows amorphous orcircular particles with no particular shape or size. The large samplesappear to have small circular structures, which might represent pores,and a topographical surface. As a whole the material is unordered andlacks the mesopores seen in the synthesis of porous organosilicates andcarbon compounds.

As shown in FIG. 3, both MC-A and MC-B have similar responses on cyclicvoltammetry peaking around −0.5 and 0.2 volts. Without any otherchemicals present EPOP shows oxidative and reductive potential at lowvoltage, nearing a milliamp at negative voltages. Unlike exhaustiveoxidation and reduction reactions as sequential passes are made on thesample the current increases, stepwise, until the peak current isobtained. The sample remained capable of returning to peak current evenafter seventeen cycles. The precursor generations of PAMAM showsubstantially no significant electrical activity.

The physical differences of MC-A and MC-B, based on the TEM images andthe electrochemical similarity of the two samples, could possibly be adifference in the average particle size of the material. The secondaddition of G0 to MC-A provides linkages between molecules that havealready grown to a significant size. It is possible that by modifyingthe conditions of the final step that average particle size could becontrolled. PAMAM generations are grown at cold temperatures (below 25°C.), and a room temperature step (25° C.) could easily produce lessordered particles.

It is also possible that an ordered structure was not formed because ofan incompatibility of the PAMAM dendrimers and the liquid-crystaltemplating method used. EPOP is completely insoluble in polar organicsolvents, and soluble in water. Acetone, which is a compatible solventfor Pluronic P123, caused EPOP to aggregate and clump. It is thereforereasonable to assume that a similar interaction may exist with P123,making it impossible to coordinate around the template. It is possiblethat a different templating method would make it possible to coordinatethe structure of the material. Further attempts at synthesis, couldattempt to control these variables.

Regardless of the structure of the material, it is remarkablyelectroactive and can function as a sensor as shown in FIGS. 4-12. Noother PAMAM related synthesis uses the polymer as the substance that iselectroactive.

The claims are not meant to be limited to the materials and methods,examples, and embodiments described herein.

The invention claimed is:
 1. A chemical composition comprising: